The seven E. coli ribosomal RNA operon upstream regulatory regions differ in structure and transcription factor binding efficiencies

Ribosomal RNAs inE. coliare transcribed from seven operons, which are highly conserved in their organization and sequence. However, the upstream regulatory DNA regions differ considerably, suggesting differences in regulation. We have therefore analyzed the conformation of all seven DNA elements located upstream of the majorE. colirRNA P1 promoters. As judged by temperature-dependent gel electrophoresis with isolated DNA fragments comprising the individual P1 promoters and the complete upstream regulatory regions, all seven rRNA upstream sequences are intrinsically curved. The degree of intrinsic curvature was highest for therrnBandrrnDfragments and less pronounced for therrnAandrrnEoperons. Comparison of the experimentally determined differences in curvature with programs for the prediction of DNA conformation revealed a generally high degree of conformity. Moreover, the analysis showed that the center of curvature is located at about the same position in all fragments. The different upstream regions were analyzed for their capacity to bind the transcription factors FIS and H-NS, which are known as antagonists in the regulation of rRNA synthesis. Gel retardation experiments revealed that both proteins interact with the upstream promoter regions of all seven rDNA fragments, with the affinities of the different DNA fragments for FIS and H-NS and the structure of the resulting complexes deviating considerably. FIS binding was non-cooperative, and at comparable protein concentrations the occupancy of the different DNA fragments varied between two and four binding sites. In contrast, H-NS was shown to bind cooperatively and intermediate states of occupancy could not be resolved for each fragment. The different gel electrophoretic mobilities of the individual DNA/protein complexes indicate variable structures and topologies of the upstream activating sequence regulatory complexes. Our results are highly suggestive of differential regulation of the individual rRNA operons.

Dissecting the Mechanism of Endogenous GATA-2 Transcription: GATA Factor Interplay at Multiple Upstream Regulatory Regions Establishes Long-Range Transcriptional Control.

Given the simplicity of the DNA sequence that mediates binding of GATA transcription factors, GATA motifs reside throughout chromosomal DNA. However, analyses of GATA-1 occupancy by chromatin immunoprecipitation (ChIP) revealed that GATA-1 discriminates exquisitely among these sites. GATA-2 selectively occupies the −2.8 kb region of the GATA-2 locus in the active state, despite there being numerous GATA motifs throughout the locus. The GATA-1-mediated displacement of GATA-2 is tightly coupled to repression of GATA-2 transcription. We have used high-resolution ChIP to show that GATA-1 and GATA-2 occupy two additional regions, −3.9 kb and −1.8 kb of the GATA-2 locus. GATA-1 and GATA-2 had distinct preferences for occupancy at these regions, with GATA-1 and GATA-2 occupancy highest at the −3.9 kb and −1.8 kb regions, respectively. Activation of an estrogen receptor fusion to GATA-1 (ER-GATA-1) induced similar kinetics of ER-GATA-1 occupancy and loss of GATA-2 occupancy at the sites. As the distinct preferences for GATA factor occupancy were not evident from electrophoretic mobility shift assay analysis in vitro, establishment of the preferences requires the native nucleoprotein structure of the GATA-2 locus. In the transcriptionally active state, the −3.9 kb and −1.8 kb regions formed strong DNaseI hypersensitive sites (HSs), and the −2.8 kb region formed a weak HS. Whereas ER-GATA-1-instigated repression abolished the −1.8 kb HS, the −3.9 kb HS persisted in the inactive state. Transient transfection analysis provided evidence that the −3.9 kb region functions distinctly from the −2.8 kb and −1.8 kb regions. Molecular studies, including capturing chromosome conformation (3C) analysis, are underway to dissect the structure/function of the upstream regulatory regions. Based on the high sequence conservation and occupancy by GATA factors in vivo, targeted deletions of the −2.8 kb and −1.8 kb regions were generated. Mutant mice are being developed, and functional analysis is being conducted via in vitro differentiation of homozygous mutant ES cells. Our results support a model in which dynamic changes in GATA factor occupancy and chromatin structure at the −3.9 kb, −2.8 kb, and −1.8 kb regions are intimately controlled by Friend of GATA-1 and additional coregulators. This GATA factor interplay is essential for GATA-2 transcriptional regulation and therefore the control of hematopoiesis.